ARTICLE pubs.acs.org/JAFC
Chondroprotective Role of Sesamol by Inhibiting MMPs Expression via Retaining NF-KB Signaling in Activated SW1353 Cells Yung-Chang Lu,†,§ Thanasekaran Jayakumar,§ Yeh-Fang Duann,# Yung-Chen Chou,§ Cheng-Ying Hsieh,§ Shin-Yun Yu,§ Joen-Rong Sheu,§,* and George Hsiao*,§ †
Department of Orthopaedic Surgery, Mackay Memorial Hospital, Taipei, and Department of Leisure Sports and Health Management, College of Humanities and Sciences, St. John's University,Tamsui, Taipei. Taiwan § Graduate Institute of Clinical Medicine, Department of Pharmacology, Taipei Medical University, Taipei, Taiwan # Department of Chemical Engineering and Biotechnology and Institute of Chemical Engineering, National Taipei University of Technology, Taipei, Taiwan ABSTRACT: Overexpression of matrix metalloproteinases (MMPs) is a major pathological factor causing cartilage destruction in osteoarthritis (OA). This study aimed to investigate the effects and mechanisms of sesamol on expression of MMPs in activated chondrosarcoma cells. Sesamol significantly attenuated TNF-R- and IL-1β-induced gelatinolysis and expression of MMP-9 in a concentration-dependent manner in SW1353 cells. Additionally, both MMP-1 and -13 stimulated by PMA were inhibited by sesamol. On the other hand, the NF-κB signaling activation through IκB-R degradation was restored by sesamol under TNF-R or PMA stimulation. Furthermore, this bioactive compound exerted the reduction on phosphorylation of ERK1/2 or p38 MAPKs after either PMA or IL-1β stimulation. This study also evaluated whether sesamol down-regulates MMP expression in the joint cartilage of monosodium iodoacetate (MIA)-induced OA in rats. Sesamol prevented the expression of MMP-1 and -9 in the cartilage of MIAinduced OA in rats. The results of this study demonstrate that sesamol inhibits cytokine- or PMA-induced MMPs expression through the signal pathways of either NF-κB or ERK/p38 MAPKs down-regulation. This study also showed that sesamol attenuates destructive factor expression in vivo, providing a potential strategy for the chondroprotective therapy in OA. KEYWORDS: human chondrosarcoma cells, sesamol, TNF-R, IL-1β, PMA, MMPs, MAPKs, IκB-R
’ INTRODUCTION Osteoarthritis (OA) is the most common form of joint disease and is widespread in the elderly population;1 it affects the weightbearing joints (knees, hips) and is associated with degeneration of the articular cartilage and subchondral bone. Cartilage resorption is also a hallmark of osteoarthritis, although the pathogenic processes are quite different. A number of mechanisms are thought to be concerned in the destruction of cartilage, which can be categorized as intrinsic or extrinsic. Intrinsic resorption takes place when chondrocytes, the only cells present in the articular cartilage, exert their capacity to affect extracellular matrix resorption. Under the influence of cytokines, in particular, tumor necrosis factor (TNF)-R, interleukin (IL)-1, and IL-6, these cartilage cells switch into a catabolic mode and degrade the surrounding extracellular matrix.2 In contrast, extrinsic resorption is mediated by tissues or cells that lie outside the articular cartilage. The matrix metalloproteinases (MMPs) are a family of enzymes that facilitate cartilage turnover and breakdown; their levels are elevated in joint tissues of patients with rheumatoid arthritis (RA) and OA.3,4 These proteolytic enzymes attack and degrade components of the extracellular matrix. Importantly, they contribute to collagen type II and other matrix protein breakdown. Inflammatory cytokines, such as IL-1β and TNF-R, have been reported to stimulate inducible expression of MMPs (1, 3, 9, and 13) in cartilages.5 Among the MMPs, collagenases are predominantly important because of their ability to cleave fibrillar collagen, which is the most abundant component of the extracellular matrix.6 MMP-1 (collagenase-1) is expressed ubiquitously and is found in r 2011 American Chemical Society
various cells, including chondrocytes.7 MMP-13 (collagenase-3) has long been regarded as the major source of collagen degrading activity, because it has preferential capacity to degrade type II collagen.8 Phorbol 12-myristate 13-acetate (PMA), as a protein kinase C (PKC) activator, was reported to be involved in the expression of MMPs in activated human chondrocytes.9 In response to IL-1β and TNF-R, a human chondrosarcoma cell line, SW1353, has been demonstrated to serve as a model that is compatible with primary chondrocytes in OA.9 It is also well documented that IL-1 and TNF-R are capable of activating the mitogen-activated protein (MAP) kinase family including extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinases, and c-Jun N-terminal kinase (JNK) in human chondrocytes.10 IκBR is reported to play a significant role in patients with arthritis due to its inhibition of MMP-1 and MMP13 production.11 Numerous inhibitors of the MMPs have been proposed as potential therapeutic agents, and the various types of compounds and their activities have been reviewed.12 Administration of chondroprotective substances can be used to counteract and/or block the actions of pro-inflammatory cytokine. Sesame (Sesamum indicum Linn., Pedaliaceae) has been categorized as a traditional health food in India and other East Asian countries.13 Sesamol is the major constituent of sesame Received: December 7, 2010 Revised: February 22, 2011 Accepted: February 28, 2011 Published: March 23, 2011 4969
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Journal of Agricultural and Food Chemistry seed oil; it is more resistant to oxidative deterioration than other vegetable oils.14 Sesamol has been shown to possess neuroprotective,15 hepatoprotective,16 anti-inflammatory,17 chemopreventive,18 and antiaging properties.19 A study has also reported that sesamin inhibits lipopolysaccharide-induced cytokine production by suppression of p38 mitogen-activated protein kinase and nuclear factor-κB.20 In our recent study, we found that sesamol strongly attenuated the signaling of platelet and its aggregation.21 A recent study has also demonstrated the upregulation of MMPs in knee cartilage from monosodium iodoacetate (MIA)-injected rats and inhibition of the degenerative changes by several MMP inhibitors.22 Hitherto, research has tended to focus on cytokine-induced expression of MMPs and their suppression by some natural compounds on chondrocytes in protecting of cartilages; however, there is relatively no information about the inhibitory mechanisms of sesamol on inducible MMPs in chondrosarcoma cell lines, SW1353, in terms of protecting cartilage lesion. Hence, the present study was undertaken to investigate the effects of sesamol on MMPs in chondrosarcoma cell lines, SW1353, under IL-1β, TNF-R, and PMA stimulation and on MIA-induced OA in the rat animal model.
’ MATERIALS AND METHODS Materials. Sesamol, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium dodecyl sulfate (SDS), phenylmethanesulfonyl fluoride (PMSF), leupeptin, aprotinin, sodium fluoride, sodium orthovanadate, sodium pyrophosphate, diethyl pyrocarbonate (DEPC), phorbol 12-myristate 13-acetate (PMA), and bovine serum albumin (BSA) were all purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human TNF-R was from Pepro Tech EC (London, U.K.). Anti-mouse and anti-rabbit immunoglobulin G (IgG)-conjugated horseradish peroxidase (HRP) was purchased from Amersham Biosciences (Sunnyvale, CA) and/or Jackson-ImmunoResearch (West Grove, PA). A mouse monoclonal antibody (mAb) specific for human native 92 kDa MMP-9 was purchased from LabVision/NeoMarkers (Fremont, CA). A rabbit polyclonal antibody (pAb) specific for IκBR was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-p38 MAPK and anti-phospho-c-Jun N-terminal kinase (JNK) (Thr183/Tyr185) mAbs, and the anti-phosphop44/p42 extracellular signal-regulated kinase (ERK) (Thr202/Tyr204) polyclonal antibody were from Cell Signaling (Beverly, MA); the Hybond-P PVDF membrane, ECL Western blotting detection reagent, and analysis system were from Amersham (Buckinghamshire, U.K.). All other chemicals used in this study were of reagent grade. Sesamol was dissolved in 0.5% dimethyl sulfoxide (DMSO) and stored at 4 °C until used in in vitro studies. Cell Cultivation. Human chondrosarcoma cells, SW1353, were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in Ham’s F-12 and DMEM (1:1) supplemented with Lglutamine (3.65 mM), penicillin (90 units/ml), streptomycin (90 μg/ mL), HEPES (18 mM), NaHCO3 (23.57 mM), and 10% heat-inactivated fetal bovine serum (FBS) at 37 °C in humidified air with 5% CO2. Stimulation Experiments. For stimulation of inflammatory agent and cytokines, chondrosarcoma SW1353 cells were seeded at 2.5 106 per well of Costar 6- well tissue culture plates in complete media until a confluence of 85% was reached (usually for 24 h). After 24 h, cells were changed to serum-free media. Cells were treated with sesamol (520 μM) for 15 min after 24 h of changing serum-free media and then treated with PMA (10 ng/mL), TNF-R (30 ng/mL), and IL-1β (30 ng/mL) for another 24 h. At the end of the incubation period, conditioned medium and cell supernatants were collected and stored at 80 °C for gelatin zymography and Western blotting assay, respectively.
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Viability Assay. The cytotoxic effects of sesamol against the SW1353 cell line were determined by the MTT method as described previously.23 Briefly, cells (2 106 cells/mL) were incubated in 12-well plates with different concentrations (520 μM) of sesamol for 24 h at 37 °C. At 22 h, the MTT solution was added to each well as a final concentration of 0.5 mg/mL. After 2 h incubation at 37 °C, the supernatant was discarded and replaced with DMSO to dissolve the formazan product, which was measured at 550 nm in a spectrophotometric plate reader. The following formula was used to calculate the percentage of cell viability: percentage cell viability = (absorbance of the experiment samples/absorbance of the control) 100%. Gelatin Zymography. MMP-9 expression was detected by gelatin zymography as described by Chung et al.24 The conditioned medium was mixed with nonreducing buffer (500 mM Tris-HCl, 25% glycerol, 10% SDS, and 0.32% bromophenol blue; pH 6.8) and electrophoresed in 10% polyacrylamide gel containing gelatin (1 mg/mL). After electrophoresis, the gels were washed two times with 2.5% Triton X-100 to remove the SDS and then incubated with reacting buffer containing 50 mM Tris-base, 200 mM NaCl, 5 mM CaCl2, and 0.02% Brij 35 (pH 7.5) for 17 h in a closed container at 37 °C. At the end of the incubation, the gels were fixed with a fixing solution (7% acetic acid and 40% methanol, v/v) for 30 min. Gels were stained with a solution of Colloidal Brilliant Blue G in 27% methanol for 30 min or longer. Finally, a destaining solution (10% acetic acid in 25% methanol) was used to adjust the clear conditions. Clear zones (bands) against the blue background indicated the presence of degradative activity of MMP-9. SDSPolyacrylamide Gel Electrophoresis (PAGE) and Western Blot Analysis. Western blot analyses were performed as previously described.23 Lysates from each sample were mixed with 6 sample buffer (0.35 M Tris, 10% w/v SDS, 30% v/v glycerol, 0.6 M DTT, and 0.012% w/v bromophenol blue, pH 6.8) and heated to 95 °C for 5 min. Proteins were separated by electrophoresis and transferred onto nitrocellulose membranes (for MMP-9) and polyvinylidene difluoride (PVDF) membranes (for MMP-1/-13, p38, pERK1/2, c-JUN, and IκB-R). The membranes were blocked with 5% nonfat milk in TBS0.1% Tween 20 and sequentially incubated with primary antibodies and HRP-conjugated secondary antibodies, followed by enhanced chemiluminescence (ECL) detection (Amersham Biosciences). BIO-PROFIL Bio-1D light analytical software (Vilber Lourmat, Marue La Vallee, France) was used for the quantitative densitometric analysis. Data of specific protein levels are presented as relative multiples in relation to the control.
Induction of Iodoacetate-Induced Osteoarthritis and Treatment in Vivo. Male Wistar rats (300450 g) were used for the experiments. All animal experiments and care were performed according to the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, 1996). All procedures were approved by the Animal Care and Use Committee, LAC-98-0036. The animals were acclimated for 1 week prior to dosing, during which time they had free access to food and water ad libitum. Eighteen such acclimated rats were divided into three groups of six each: group I, normal rats (received saline); group II, monosodium iodoacetate (MIA)induced untreated rats; group III, MIA-induced rats treated with sesamol. Arthritis was induced by a single intraarticular injection of iodoacetate into the knee joint of anesthetized rats.22 A 10 mg/mL concentration of MIA (Aldrich Chemical, Milwaukee, WI) was prepared using injectable saline as the vehicle. After appropriate anesthesia (1 mL/kg bw, of 40% chloral hydrate), each rat was positioned on their back and the left leg was flexed 90° at the knee. The patellar ligament was palpated below the patella, and the injection was made into this region. Each rat received a 25 μL intraarticular injection of MIA into the left knee using a glass gastight syringe with a 0.5 in. needle for 7 days and then was treated with sesamol dissolved in saline (30 mg/kg bw) by oral feeding twice a week for 2 weeks. After 2 weeks of sesamol 4970
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Figure 1. Effects of sesamol on tumor necrosis factor (TNF)-R-induced expression of MMP-9 in SW1353 chondrosarcoma cell line: (A) structure of sesamol; (B) cells were treated with various concentrations of TNF-R for 24 h (cell-free conditioned media were assayed for MMP-9 activation by gelatin zymography); (C) effects of sesamol on TNF-R-induced activation of MMP-9 (cells were pretreated with DMSO or various concentrations of sesamol for 15 min before treatment with TNF-R (30 ng/mL) for 24 h); (D) cell lysates were obtained and analyzed for MMP-9 protein expression by Western blotting (bottom, densitometric analysis of bands for MMP-9, relative to R-tubulin normalized to the resting condition). ###, P < 0.001 compared with the resting group; /, P < 0.05, //, P < 0.01, and ///, P < 0.001, compared with the vehicle group. treatment, animals were sacrificed, and the left joint was immediately disarticulated prior to the following analyses. Preparation of Cartilage Extracts. The tissue surrounding the joint was removed, the joint was disarticulated, and the cartilage was scraped from the surface of the tibia and femur using a scalpel blade and an illuminated magnifier. The cartilage chips were placed into a preweighed vial, the weight of the cartilage was determined, and cold extractant was added to the vial. MMPs were extracted using a two-step procedure. Ten
microliters of 0.25% Triton X-100 with 0.01 M CaCl2 was added per milligram of cartilage. Extraction was performed for 20 min at 4 °C followed by centrifugation in a microfuge for 20 min at 13000 rpm. The supernatant was removed and the pellet extracted with 50 mM Tris-HCl, 0.1 M CaCl2, 0.15 M NaCl, pH 7.5. The extract was heated for 4 min at 60 °C in a water bath and centrifugation repeated as described above. The supernatant was collected and combined with the supernatant from the Triton-X 100 extract and was utilized for Western blot analysis for MMPs as described above. 4971
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Statistical Analyses. The experimental results are expressed as the mean ( SEM and are accompanied by the number of observations. For analysis of the results, a one-way analysis of variance (ANOVA) test was performed using Sigma Stat v3.5 software. When group comparisons showed a significant difference, the StudentNewmanKeuls test was used. A P value of
